1. Field of the Invention
The present invention relates to human fertility and more particularly, to means and methods for determining uterine endometrial receptivity for a fertilized egg and a method and composition for contraception.
2. Description of Related Art
There are many issues involved with the evaluation of a woman's fertility. Production and availability of an egg, fertilization of the egg, and other issues involving the physiology of the egg are involved. Another general issue is the nature of the receptivity of the endometrium. If not receptive to implantation, implantation of a fertilized egg may not occur or may occur in an abnormal manner. If receptive, implantation is optimized. Accordingly, it would be useful to have a diagnostic tool for determining endometrial receptivity.
Reproductive Failure and Assisted Reproductive Technologies: In 1975, Roberts and Loewe, using a mathematical equation, suggested that 78% of fertilizations fail to result in a live birth [Roberts and Loewe, 1975]. Lindley [1979] estimated that only about 30% of all conceptions survive to birth, 15% end in recognizable miscarriage, and the other 55% are lost in the early stages of pregnancy. Inadequate uterine receptivity and subsequent embryo implantation failure, rather than fertilization failure, has been implicated as the crucial event which differentiates fertile and non-fertile ovulatory cycles [Navot et al., 1989]. The importance of endometrial adequacy and receptivity has become even more apparent with the evolution of the assisted reproductive technologies. In vitro fertilization and embryo transfer procedures produce fertilization rates of 70 to 90% whereas pregnancy rates after embryo transfer remain disappointing low ranging from 15 to 25% [Edwards, 1985; Cohen, 1991]. Ovarian hyperstimulation protocols used for these procedures have been associated with several factors that may contribute to lower implantation rates including production of oocytes and subsequent embryos that are chromosomally unbalanced, creation of embryos that are biochemically defective and alteration of the maternal uterine environment [Angell et al., 1987; O'Neill et al., 1987; Collier et al., 1989; Safro et al., 1990]. An improved knowledge of factors influencing maternal uterine environment and human embryo implantation may help "save" these embryos and reduce embryo loss.
Studies have demonstrated that ovarian hyperstimulation impedes implantation by causing adverse changes in uterine receptivity [Fossum et al., 1989; Paulson et al., 1990; Sharma et al., 1990]. For example, clomiphene citrate, an antiestrogen widely used for ovarian hyperstimulation, has been linked to lower conception rates, a higher incidence of spontaneous abortion and might be partially responsible for deficiencies in endometrial development [Jones et al., 1970]. Clomiphene citrate inhibits decidual induction in pseudopregnant rats when administered prior to pyrathiazine injection (induces decidualization in rats) and inhibits implantation of rat blastocysts when administered at the time of their adherence to the uterus [Barkai et al., 1992]. This inhibition could not be explained on the basis of the current understanding of mechanisms of estrogen action. Fazleabas et al. [1991] demonstrated that clomiphene citrate markedly decreased endometrial .alpha..sub.2 -pregnancy associated endometrial globulin (.alpha..sub.2 PEG; also called placental protein 14 or PP14 or progesterone dependent endometrial protein, PEP) production in vitro. Arthur and colleagues [1995] have shown that PP14 concentrations are depressed and insulin-like growth factor binding protein-1 (IGFBP-1 originally called .alpha..sub.1 PEG or PP12) concentrations are elevated in pregnancies that follow down-regulation of the anterior pituitary (Buserelin, Hoechst, Hounslow, UK) and exogenous hormone support (Pergonal, Serono, Welwyn Garden City, UK) prior to a frozen-thawed embryo transfer. No correlation could, however, be found between hCG, IGFBP-1, progesterone and PP14 concentrations suggesting no primary associations between these compounds.
The above studies demonstrate that ovarian hyperstimulation/ovulation induction protocols alter normal endometrial development and protein synthesis. Unfortunately, no noninvasive methods or markers have yet been developed to detect the effects of these protocols on subsequent uterine receptivity. An accurate marker for uterine receptivity for blastocyst implantation is urgently needed as a clinical adjuvant in assisted reproduction procedures and may improve the pregnancy rate in women who have experienced difficulty in conceiving due to an anomalous state of uterine receptivity and implantation failure.
The efficiency of implantation in assisted reproduction procedures in quite low. Presently, three to four high quality cleavage stage embryos are transferred to the uterus following in vitro fertilization. Routinely, such embryo transfers result in a singleton pregnancy in approximately 15 to 25% of the patients. Higher risk, multiple gestations do result from these transfers. Improved knowledge of factors influencing human embryo implantation and an accurate marker of uterine receptivity would help reduce embryo loss by reducing the numbers of embryos needed for transfer and reducing the potential for multiple gestation. The need for fewer embryos may also reduce the amount of exogenous ovarian stimulation needed and thereby reduce risks associated with taking these compounds. The need for reduced numbers of embryos may be most important in reproductively older women (&gt;38 years of age) who frequently are approaching the menopause and are producing fewer quality ooctyes and consequently fewer embryos.
An accurate marker would permit monitoring of the state of uterine receptivity prior to embryo transfer. Embryo transfer procedures could be timed to coincide with a receptive endometrium or delayed, cryopreserving embryos, until a more appropriate state of receptivity was attained. This may be especially relevant in patients where ovarian hyperstimulation and ovulation induction has altered the natural course of endometrial development. A marker of uterine receptivity may also be diagnostic in women who repeatedly fail to become pregnant and whom have no other apparent etiology for their infertility.
An accurate marker for uterine receptivity may also help reduce the cost of infertility procedures. The cost of assisted reproductive procedures varies across the United States (estimated ranges from $10,000 to $25,000 and higher for an in vitro fertilization/embryo transfer procedure). Often, infertility procedures are not covered by insurance. The most expensive portion of infertility therapies is usually the drugs. As stated above, if uterine receptivity can be defined by a marker and fewer embryos are needed, then less drug may also be needed thereby reducing the cost. Inadequate uterine receptivity could be diagnosed and possibly therapeutically enhanced prior to attempting or during assisted reproduction procedures thus requiring fewer overall attempts per conception.
The composition of uterine secretions is of considerable interest because of how they may effect reproductive processes such as sperm migration, embryo transport and implantation in the uterine endometrium. Uterine fluid, as it exists in vivo, is a mixture of proteins synthesized and secreted by the endometrium, proteins transferred across the endometrium from the blood stream or adjacent cells and proteins from the oviduct and/or cervix. As a result of progesterone (P) stimulation, the secretory stage human endometrium synthesizes and secretes specific proteins including pregnancy-associated endometrial .alpha..sub.1 -globulin (.alpha..sub.1 -PEG) pregnancy-associated endometrial .alpha..sub.2 -globulin (.alpha..sub.2 -PEG), prolactin (PRL) and progesterone-induced uterine protein-1 (PUP-1)[Seppela et al., 1992; Maslar and Riddick, 1979; Sharpe et al., 1993]. Although .alpha..sub.1 -PEG has been described by different names in the literature including placental protein 12 (PP12) and endometrial protein 14 (EP14), subsequently analysis has identified this protein as insulin-like growth factor binding protein-1 (IGFBP-1) [Koistinen et al., 1992].
Preimplantation development and communication: embryo/endometrium cross-talk: Factors secreted by sheep and cattle conceptuses that are structurally related to interferons (IFNs) have been identified (ovine trophoblastic protein-1 and bovine trophoblastic protein-1) and implicated as mediators of maternal recognition of pregnancy [Godkin et al., 1984; Lifsey et al., 1989]. The fact that preimplantation conceptuses of other species, e.g. pig, release substances with antiviral activity suggests the IFNs may play a role in pregnancy that extends beyond domestic ruminants [Roberts, 1989; Roberts et al., 1990]. Embryos also produce platelet activating factor (PAF) which is capable of inducing transient maternal thrombocytopenia [O'Neill et al., 1985]. Embryo PAF is being evaluated as a means of monitoring pre- and post-implantation embryo viability following in vitro fertilization. Two gelatinases (also called matrix metalloproteinase: MMP2 and MMP9) produced by porcine embryos during the preimplantation period may be involved in the dramatic changes in endometrial morphology that occur during this period [Chamberlin and Menino, 1995]. Despite this accumulation of knowledge, surprisingly scant information about mechanisms controlling the receptive phase of the uterine endometrium and subsequent blastocyst implantation is available.
For more than two decades it has been known that synchronized development of the preimplantation embryo and hormonal preparation of the uterus to the receptive state were essential for initiation of pregnancy and subsequent decidualization of the uterine stroma [Psychoyos, 1973]. Glandular and luminal endometrial epithelia require estrogen while endometrial stroma requires both estrogen and progesterone for proliferation and differentiation. More recent concepts have emerged including mediation of estrogen and progesterone action in the endometrium by specific growth factors, growth factor receptors, cytokines, enzymes and their inhibitors [Giudice, 1994; Smith, 1994; Tabibzadeh, 1994; Rodgers et al., 1994]. For example, studies have documented expression of matrix metalloproteinases (MMPs) and their inhibitors in the human endometrium [Rodgers et al., 1994; Hampton and Salamonsen, 1994; Matrisian et al., 1994]. These studies have demonstrated the requirement for a balance between the expression of MMPs and their inhibitors in the continual processes of growth, differentiation and destruction of the endometrium that occur throughout the menstrual cycle. Production of MMPs by both the embryo and the endometrium suggests a possible mechanism of cross-talk and regulation of the process of implantation by these two entities.
Uterine protein content increases during the preimplantation period in most mammalian species. These increases are due to decreasing water content and increasing viscosity due to the influence of progesterone. Progesterone also stimulates the synthesis and secretion of specific endometrial proteins in several mammalian species including the human [i.e., Mulholand and Villie, 1984; Sharpe et al., 1991; Sharpe and Vernon, 1993; Sharpe et al., 1993]. The functions of some of these proteins such as uteroglobin from the rabbit endometrium (immunomodulation of fetomaternal interactions) and uteroferrin in the pig (placental iron transport) have been defined [Beier, 1968; Roberts and Bazer, 1980]. In the human, .alpha..sub.1 -PEG has been identified as insulin-like growth factor (IGF-I) binding protein while the sequence of .alpha..sub.2 -PEG indicates homology to .beta.-lactoglobulin (up to 53%) and human retinol binding protein (23%) and may serve an immunoregulatory role or as a transfer protein [Seppala et al., 1987; Seppala et al., 1991]. Endometrial prolactin (PRL) production is also stimulated by progesterone in the human uterus [Maslar and Riddick, 1979]. Disappointingly, and for various reasons, none of these proteins has yet been proven useful for prediction of uterine receptivity and successful embryo implantation.
Messenger RNA encoding human IGFBP-1 and corresponding protein has been identified in secretory stage endometrial stromal cells but not secretory stage epithelial cells nor any proliferative stage endometrial cells [Julkunen et al., 1990; Wahlstrom et al, 1984]. Studies of the amino acid sequence of these immunologically indistinguishable proteins have demonstrated significant sequence homology with .beta.-lactoglobulins [Julkunen et al., 1990].
Immunolocalization of .alpha..sub.2 -PEG, PP14 and PEP has been demonstrated in secretory stage glandular epithelium but not the stroma [Sharpe et al., 1993; Joshi et al., 1980, 1986]. Prolactin is synthesized by P-stimulated endometrial stromal cells and immunolocalizes in subpopulations of late secretory stage decidualized stromal cells and in epithelial cells [Maslar and Riddick, 1989; Daly et al., 1983; McRae et a. 1986]. While, these are known, there is no clinical utility for these proteins that would serve as a marker for endometrial receptivity and therefore fertility or infertility.
Therefore it is desirable to obtain means and a method for determining endometrial receptivity based on unique proteins synthesized and secreted by human endometrium in vitro and in vivo.
Improved methods of contraception, that is prevention of fertilization or implantation of the fertilized egg, are needed particularly in light of increasing population pressure. Many efforts have been made to provide improved contraception utilizing devices or hormonal therapy for females as for example as set forth in U.S. Pat. Nos. 5,771,900; 5,756,115; 5,583,129; 4,922,928; 4,703,752; and 4,564,362 and the references cited therein. However, they are not always successful in providing contraception and improved methods are needed. Progesterone receptor antagonists (such as RU486) alter uterine biochemistry but this alteration is used to induce abortion or as a morning-after pill to prevent implantation. It would be useful to have other methods available which can change uterine receptivity biochemcially.